Serotonin reuptake inhibitors as drugs having peripheral-system-restricted activity

ABSTRACT

Serotonin reuptake inhibitor compounds which are designed to exert serotonin uptake inhibitory activity in the peripheral system while being devoid of CNS activity, and a process of preparing same are disclosed. Further disclosed are pharmaceutical compositions containing same and uses thereof in the treatment of medical conditions associated with peripheral serotonin levels and/or activity, and/or platelet aggregation.

RELATED APPLICATIONS

This application is a continuation-in-part (CIP) of U.S. patentapplication Ser. No. 12/308,671 filed on Jun. 29, 2010, which is aNational Phase of PCT Patent Application No. PCT/IL2007/000756 filed onJun. 21, 2007, which claims the benefit of priority under 35 USC §119(e)of U.S. Provisional Patent Application No. 60/815,582 filed on Jun. 22,2006. The contents of the above applications are all incorporated byreference as if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel therapeutically active compoundsand more particularly, to novel serotonin reuptake inhibitors (SRI) andtheir use as therapeutic agents for treating platelet-aggregationassociated diseases and disorders, as well as other peripheral medicalconditions.

Acute myocardial infarction (AMI or MI), also known as a heart attack,is a disorder that occurs when the blood supply to a part of the heartis interrupted, and the resulting ischemia (shortage in oxygen) causesdamage and potential death of heart tissue. Currently MI is the leadingcause of death for both men and women all over the world, causing 12.6%of deaths worldwide (a higher mortality rate than cancer). According tothe World Health Organization reports, rates of MI-related death aremuch higher in countries with higher life expectancies, and keepincreasing as better treatments become available for common cancers suchas colon and breast cancers. Some of the main risk factors are aprevious history of vascular disease such as atherosclerotic coronaryheart disease and/or angina, a previous heart attack or stroke, anyprevious episode of abnormal heart rhythms or syncope, age (men over 40and women over 50), smoking, excessive alcohol consumption, the abuse ofcertain illicit drugs, high triglyceride levels, high LDL and low HDL,diabetes, high blood pressure, obesity, and chronically high levels ofmental stress.

The risk of a new or recurrent myocardial infarction decreases withstrict blood pressure management and lifestyle changes, primarilysmoking cessation, regular exercise, a sensible diet for patients withheart disease, and limitation of alcohol intake. Patients whomexperienced a coronary event are typically prescribed several chronicmedications with the aim of preventing secondary cardiovascular eventssuch as further myocardial infarctions, congestive heart failure orcerebrovascular accident (CVA). Such medications include anti-plateletdrugs such as aspirin and/or clopidogrel (Plavix) which reduce the riskof plaque rupture and recurrent myocardial infarction.

Ischemic heart disease or coronary artery disease (CAD), stroke, orpulmonary embolism, are all diseases that share the phenomena ofspontaneous formation of small blood clots, unrelated to injury of bloodvessels and initiated by aggregation of blood platelets inside intactblood vessels. These small platelet aggregates are carried in the bloodstream until they reach small capillaries, where they might cause localischemia by blocking small capillaries in the heart, lungs or braintissues. Such tiny clots are also implicated in the blockade of bloodvessels in the legs, a common complication of diabetes. Hence, drugsthat can treat ischemia can be used beneficially to treat medicalconditions which include ischemic heart disease (IHD), myocardialinfarction (MI), cerebral stroke, pulmonary embolism, and type-2diabetes-associated vascular abnormalities.

Aspirin, introduced in the late 19^(th) century by Bayer (Germany), isthe first synthetic drug ever marketed as a pain-relief andanti-inflammatory drug. In the second half of the 20^(th) centuryaspirin has been shown to act by inhibiting the activity ofcyclooxygenase enzymes, a key step in the synthesis of prostaglandins,endogenous mediators of pain and inflammation. It was realized thatsince prostaglandins play a key role in the increased plateletaggregation associated with inflammation, aspirin can act as a potentanti-platelet agent when given chronically, and therefore can protectfrom diseases such as CAD, MI, stroke and pulmonary embolism. Thus,aspirin has become the most widely prescribed drug globally, and isgiven as preventive treatment in CAD [1, 2]. Aspirin is also prescribedas preventive treatment against cardiovascular complications in type-2diabetes [3].

However, blocking prostaglandins production by aspirin cannot fullyprotect individuals from increased platelet aggregation, mainly since ithas been found that additional endogenous modulators (such as, forexample, thrombin and adenosine) are implicated in platelet aggregationduring inflammation. Moreover, aspirin is contra-indicated inindividuals with ulcers, gastritis, ulcerative colitis, due to itstendency to increase gastrointestinal bleeding. Therefore, there isclinical validity to develop additional anti-platelet drugs, as add-ontherapy for individuals who receive chronic aspirin, or as replacementfor aspirin in individuals in whom aspirin is contra-indicated.

Clopidogrel causes irreversible inhibition of the adenosine diphosphate(ADP) receptor (P2Y12) on platelet cell membranes, which is a keyparticipant in the process of platelet aggregation. The obturation ofthis receptor inhibits platelet aggregation by blocking activation ofthe glycoprotein IIb/IIIa pathway. Clopidogrel is indicated forprophylactic prevention of vascular ischaemic events in patients withsymptomatic atherosclerosis, in cases of acute coronary syndrome withoutST-segment (the part of an electrocardiogram immediately following theQRS complex and merging into the T wave) elevation (NSTEMI) along withaspirin, and for the prevention of thromboembolism after placement ofintracoronary stent also along with aspirin. The use of clopidogrel overaspirin is recommended for patients with a history of gastric ulcerationrequiring anti-platelet therapy. However, a recent study has shown thatpatients with healed aspirin-induced ulcers receiving aspirin plus theproton pump inhibitor esomeprazole had a lower incidence of recurrentulcer bleeding than patients receiving clopidogrel. Additionally,antithrombotic doses of clopidogrel were found to have limited effectson bleeding and standard measures of platelet aggregation [4].Furthermore, clopidogrel is associated with other serious adverseeffects which include severe neutropenia, thrombotic thrombocytopenicpurpura, hemorrhage which is aggravated by the co-administration ofaspirin, gastrointestinal hemorrhage, cerebral hemorrhage and erectiledysfunction.

Serotonin (5-HT) is a major central nervous system (CNS) monoamineneurotransmitter, which is synthesized in serotonergic neurons in theCNS (about 10%) and enterochromaffin (EC) cells (Kulchitsky cells) inthe gastrointestinal tract of animals (about 90%). Serotonin is alsofound in many mushrooms and plants. Serotonin is stored outside thebrain mostly in platelets of the blood stream. Serotonin was firstisolated and named in 1948 by Rapport, Green and Page, who identified itinitially as a vasoconstrictor substance in blood serum—hence serotonin,a serum agent affecting vascular tone. Rapport and co-workers alsoidentified serotonin chemically as 5-hydroxytryptamine (5-HT), and sincethen it was studied and found to exhibit a wide range of physiologicalroles.

Most of 5-HT is synthesized by tryptophan hydroxylase-1 (TPH1), which isexpressed almost exclusively in the enterochromaffin cells of thegastrointestinal tract. 5-HT is secreted into the blood and taken up bythe 5-HT transporter (5-HTT) primarily into platelets, which are therichest reservoir of 5-HT in the periphery. In the blood, 5-HT is storedalmost exclusively in the dense granules of platelets and is almostabsent in the plasma. Notably, blood lymphocytes (both B and Tlymphocytes) also express functional 5-HTT and can accumulate 5-HT.However, the capacity of lymphocytes to store and to release 5-HT hasnot been proven to date; the lymphocyte reservoir is apparently smallercompared with the large platelets 5-HT storage capacity.

As a CNS active substance, serotonin plays an important role in theregulation of aggression, mood, body temperature, sleep, vomiting,sexual drive and appetite. Low levels thereof or low bioavailability areassociated with several disorders such as increased aggressive and angrybehaviors, clinical depression, obsessive-compulsive disorder (OCD),migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia (FM orFMS), bipolar disorder, anxiety disorders and intense religiousexperiences. In addition, abnormal serotonergic neurons have beenassociated with the risk of sudden infant death syndrome (SIDS). Whentaken orally, 5-HT does not pass into the serotonergic pathways of theCNS because it cannot cross the blood-brain barrier (BBB).

In addition to its CNS activities, 5-HT is involved in severalperipheral activities; these include cardiovascular modulating effects(both vasoconstrictor and vasodilator), potent pro-thrombotic activity,endothelial mitogenic action, as well as immune modulating effects.

One method for modulating peripheral 5-HT levels is the chronic use ofserotonin-selective reuptake inhibitor drugs (SSRIs) such asalaproclate, dapoxetine, etoperidone, citalopram, escitalopram,fluoxetine, fluvoxamine, paroxetine, sertraline and zimelidine. Of notehowever, all the above-mentioned are known to penetrate the BBB readilyand thus have CNS activities in addition to their peripheral activitieson modulating 5-HT levels. Indeed the SSRIs are routinely used fortreating CNS conditions such as clinical depression,obsessive-compulsive disorder, and additional mood disorders and arehence known to belong to the class of antidepressant drugs.

Stemming from the vast research on serotonin it transpired that blockingthe 5-HT transporter (5-HTT) activity in the periphery by SSRI drugsleads to decreased platelet 5-HT storage capacity, and thereby leads toreduced biological availability of platelet 5-HT. This in turn leads toreduced platelet aggregation during inflammation.

Several studies, including use of transgenic mice deficient inperipheral 5-HT synthesis, have suggested that reducing peripheral 5-HTlevels may be beneficial for coronary artery disease (CAD) patients. Inother words, reducing platelets 5-HT storage capacity is reflected inanti-thrombotic consequences. Indeed, epidemiological studies haveindicated that patients who are treated chronically with SSRI drugs areless likely to suffer from MI and CAD [5-8].

Among the lines of evidence pointing towards potential beneficialeffects of reduced peripheral 5-HT, is the observation that transgenicmice deficient in TPH1 (tph1−/−) and therefore in 5-HT synthesis exhibitreduced thrombosis risk [9].

There are several clinical studies showing that patients who take SSRIdrugs chronically are less likely to develop thrombosis-relateddisorders, most notably, MI, stroke and CAD [7, 8, 10]. A randomizedstudy compared sertraline (marketed under the name Zoloft as well asmany other trade names) versus placebo in depressed post-acute coronarysyndromes (ACS) patients, administered in addition to the standardanti-platelet agents aspirin and clopidogrel [10]. In this study, plasmamarkers of platelet activation were monitored, and the results showedthat sertraline treatment was associated with substantially attenuatedrelease of platelet/endothelial biomarkers as compared to the placebotreatment.

Furthermore, a multi-center study that included 68 hospitals, whichfocused on the effects of SSRI treatment on first time myocardialinfarction (MI), indicated a protective effect: the odds ratio for MIamong current SSRI users compared with nonusers was 0.35 [7].

The largest study to date compared 1080 myocardial infarction (MI) casesand 4256 controls during a 3-year period [8]. Unlike the former studies,this study included patients receiving various SSRIs (paroxetine,fluoxetine, or sertraline) as well as non-SSRI antidepressants andtricyclic antidepressants. The study reported that overall, SSRI use wasassociated with a large reduction of MI risk (odds ratio of 0.59;meaning, a 41% reduction of MI risk during the 3-year follow-up period);such reductions were not observed for the non-SSRI antidepressants.Notably, the two anti-platelet agents in current clinical use, aspirinand clopidogrel, were reported to reduce MI risk by only 20% and 10%respectively [1].

It should be noted that the prospective beneficial effects of SSRIs forreducing MI, CAD and ischemic risk are not necessarily limited to theirdirect capacity for reducing thrombosis. An additional and attractivemechanism of action for the observed clinical benefits in chronic heartfailure (CHF) patients might reflect reduced endothelial mitogenesisfollowing platelet-endothelial adhesion, and hence, reduced restenosisat the coronary arteries [11]. Coronary restenosis occurs as a result ofexaggerated coronary endothelial mitogenesis, which might involveincreased activation of endothelial cells by adhering platelets andserotonin released by the latter. It is further established thatserotonin is mitogenic for endothelial cells.

Over the years, additional potential clinical indications for probablebeneficial effects of SSRI or other serotonin-modulating compounds fornon-psychiatric indications have been unveiled. These include chronicdisorders for which increased platelet activity has been reported andimplicated in the disease progression. This diseases and disordersinclude pulmonary hypertension, in which a 50% reduction in the risk ofdeath was noted in SSRI users [12]; restenosis, following electivecoronary stenting of native coronary arteries which was shown to bereduced by blocking the action of peripheral 5-HT [13]; rheumatoidarthritis [14], diabetes, where the SSRI drug fluvoxamine was shown toimprove hepatic glucose uptake in a dog [15], autoimmune disorders(e.g., multiple sclerosis, psoriasis), where mice lacking the 5-HTT (asituation mimicking chronically blocking the 5-HTT with an SSRI drug)were shown to be less sensitive to induction of experimental autoimmuneencephalomyelitis (EAE), a well-defined animal model of autoimmunedisease of the central nervous system mimicking features of the humandisease multiple sclerosis [16], kidney failure [17] and inflammatorybowel disease (IBD) [18].

In recent publications, a potential therapeutic application of 5-HTTinhibitors in human pulmonary arterial hypertension have also beensuggested, where it was shown that transgenic mice over-expressing 5-HTTin smooth muscle develop pulmonary hypertension, indicating thatblocking the 5-HTT with SSRI could be protective against this disorder[19].

However, currently, chronic treatment with SSRI drugs is reserved in theclinic for affective disorders (also known as mood disorders), mostnotably, depression and compulsive disorders, and is unwarranted aschronic treatment for people at risk of CAD, MI, stroke or otherischemic diseases, which forms a large segment of the population overthe age of 50 years. This is mainly due to the severe adverseCNS-related side-effects of chronic SSRI treatment, such as nausea,drowsiness or somnolence, headache, clenching of teeth, extremely vividand strange dreams, dizziness, changes in appetite, weight loss/gain,changes in sexual behavior (reduced libido), increased feelings ofdepression and anxiety which may sometimes provoke panic attacks,tremors, autonomic dysfunction including orthostatic hypotension,thoughts of suicide, depersonalization (derealization), flattenedemotions and increased aggressiveness. Out of this list of known adverseeffects, the three most frequently observed in SSRI users are decreasedlibido, flattened emotions and increased aggressiveness.

SUMMARY OF THE INVENTION

Serotonin is stored in blood platelets and is known to participate inmany processes which occur in the peripheral system and thereforeregulation of its levels and/or activity is a key step for therapy.

There is thus a widely recognized need for novel compounds, which wouldexhibit a serotonin reuptake inhibitory activity mainly in theperiphery, and hence could be utilized in the treatment of variousperipheral disorders, without entering the CNS and affecting brain 5-HTstorage and actions in the brain.

Embodiments of the present invention relate to novel compounds fortreating peripheral serotonin associated diseases and disorders, and,more particularly, to novel serotonin reuptake inhibitors and their useas therapeutic agents having a peripheral-restricted activity fortreating peripheral serotonin associated diseases and disorders.

Thus, according to one aspect of the present invention there is provideda serotonin reuptake inhibitor (SRI) compound, being modified so as toinclude at least one positively charged group, which is selected suchthat the modified SRI compound retains its charge at physiological pH,while substantially retaining its serotonin reuptake inhibitionactivity.

According to further features in preferred embodiments of the inventiondescribed below, the positively charged group is a quaternary ammoniumgroup.

According to further features in the described preferred embodiments thequaternary ammonium group has the formula:—(NR₁R₂R₃)⁺Z⁻

wherein:

Z is an organic or inorganic anion; and

R₁, R₂ and R₃ are each independently selected from the group consistingof alkyl, aryl and cycloalkyl.

According to still further features in the described preferredembodiments, R₁, R₂ and R₃ are each independently an alkyl having from 1to 4 carbon atoms.

According to still further features in the described preferredembodiments the alkyl is methyl.

According to still further features in the described preferredembodiments the positively charges group is a tertiary sulfonium group.

According to still further features in the described preferredembodiments the tertiary sulfonium group has the formula:—(SR₄R₅)⁺Z⁻

wherein:

Z is an organic or inorganic anion; and

R₄ and R₅ are each independently selected from the group consisting ofalkyl, aryl and cycloalkyl.

According to still further features in the described preferredembodiments, R₄ and R₅ are each independently an alkyl having from 1 to4 carbon atoms.

According to still further features in the described preferredembodiments the alkyl is methyl.

According to still further features in the described preferredembodiments, the SRI compound presented herein is derived from acompound selected from the group consisting of a selective serotoninreuptake inhibitor (SSRI), a serotonin-norepinephrine reuptake inhibitor(SNRI) and a serotonin-noradrenaline-dopamine reuptake inhibitor(SNDRI).

According to still further features in the described preferredembodiments, the SSRI is selected from the group consisting ofcitalopram, alaproclate, dapoxetine, etoperidone, fluoxetine,fluvoxamine, paroxetine, sertraline, venlafaxine and zimelidine.

According to still further features in the described preferredembodiments, the SNRI is selected from the group consisting ofdesvenlafaxine, duloxetine, milnacipran, nefazodone and venlafaxine.

According to still further features in the described preferredembodiments, the SNDRI is selected from the group consisting ofbrasofensine, tesofensine and nomifensine.

According to still further features in the described preferredembodiments, the SRI compound presented herein is derived fromcitalopram.

According to still further features in the described preferredembodiments, the SRI compound presented herein is N-alkyl-citalopram.

According to still further features in the described preferredembodiments, the alkyl is methyl.

According to still further features in the described preferredembodiments, the SRI compound presented herein is substantiallyincapable of modulating a serotonin level and/or activity in the CNS.

According to still further features in the described preferredembodiments, the SRI compound presented herein is substantiallyincapable of reducing a serotonin level and/or activity in the CNS.

According to another aspect of the present invention there is providedN-alkyl-citalopram.

According to another aspect of the present invention there is providedN-methyl-citalopram.

According to another aspect of the present invention there is provided aprocess of preparing the SRI compound of claim 1, the process iseffected by modifying the SRI compound so as to generate the at leastone positively charged group.

According to further features in preferred embodiments of the inventiondescribed below, the SRI compound has an amine group.

According to still further features in the described preferredembodiments, modifying the SRI compound is effected by N-alkylating orN-arylating the amine group.

According to still further features in the described preferredembodiments, modifying the SRI is effected by an alkylating agentselected from the group consisting of an alkyl-sulfonate, anaryl-sulfonate, an alkyleneimine, phosgene, an alkyl-tosylate, anaryl-tosylate, an alkyl-triflate, an aryl-triflate, an alkyl-halide, anaryl-halide, a dialkyl-sulfate, a diaryl-sulfate, an alumoxane, atrialkylaluminum and a tris(trialkylyl)aluminum.

According to still further features in the described preferredembodiments, the alkylating agent is methyl iodide.

According to another aspect of the present invention there is provided amethod of modulating a level and/or activity of peripheral serotonin(5-HT) in a subject while substantially maintaining a level and/oractivity of central serotonin in the subject, the method is effected byadministering to the subject a SRI compound as presented herein.

According to another aspect of the present invention there is provided ause of an SRI compound as presented herein, in the modulation of a leveland/or activity of peripheral serotonin (5-HT) in a subject whilesubstantially maintaining a level and/or activity of central serotoninin the subject.

According to further features in the described preferred embodiments,the method of modulating a level and/or activity of peripheral serotonin(5-HT) in a subject is for treating a medical condition in whichmodulating a level and/or activity of peripheral serotonin (5-HT) in asubject while substantially maintaining a level and/or activity ofcentral serotonin is beneficial.

According to still further features in the described preferredembodiments, the medical condition is selected from the group consistingof a cardiovascular disease or disorder, a cerebrovascular disease ordisorder, ischemic heart disease (IHD), myocardial infarction (MI),cerebral stroke, pulmonary embolism, type-2 diabetes-associated vascularabnormalities, pulmonary arterial hypertension, peripheral arterialocclusive disease, rheumatoid arthritis, an autoimmune disorder, kidneyfailure, inflammatory bowel disease, acute coronary syndrome, coronaryartery disease (CAD) and restenosis following coronary artery bypasspost-grafting.

According to yet another aspect of the present invention there isprovided a method of treating a disease or disorder in which reducing orpreventing platelet aggregation and/or platelet-endothelial interactionsis beneficial, the method is effected by administering to a subject inneed thereof a therapeutically effective amount of an SRI compound aspresented herein.

According to still another aspect of the present invention there isprovided a use of an SRI compound as presented herein, in the treatmentof a disease or disorder in which reducing or preventing plateletaggregation and/or platelet-endothelial interactions is beneficial.

According to yet another aspect of the present invention there isprovided a use of an SRI compound as presented herein, in themanufacture of a medicament for the treatment of a disease or disorderin which reducing or preventing platelet aggregation and/orplatelet-endothelial interactions is beneficial.

According to still another aspect of the present invention there isprovided a pharmaceutical composition comprising, as an activeingredient, an SRI compound as presented herein and a pharmaceuticallyacceptable carrier.

According to further features in the described preferred embodiments,the pharmaceutical composition is packaged in a packaging material andidentified in print, in or on the packaging material for use in thetreatment of a medical condition in which modulating a level and/oractivity of peripheral serotonin (5-HT) in a subject while substantiallymaintaining a level and/or activity of CNS serotonin is beneficial, asdescribed herein.

According to further features in the described preferred embodiments,the pharmaceutical composition is packaged in a packaging material andidentified in print, in or on the packaging material for use in thetreatment of a disease or disorder in which reducing or preventingplatelet aggregation and/or platelet-endothelial interactions isbeneficial, as described herein.

According to further features in the described preferred embodiments,the SRI compound is substantially incapable of modulating a serotoninlevel in the CNS of the subject.

According to still further features in the described preferredembodiments, the SRI compound is substantially incapable of increasing aserotonin level or activity in the CNS of the subject.

According to still further features in the described preferredembodiments, the SRI compound is substantially incapable of reducing aserotonin level or activity in the CNS of the subject.

According to still further features in the described preferredembodiments, reducing or preventing platelet aggregation and/orplatelet-endothelial interactions is effected while substantiallymaintaining a level and/or activity of CNS serotonin, thereby avoidingCNS-related effects.

According to still further features in the described preferredembodiments, the method of treatment, the pharmaceutical composition andthe uses of the SRI presented herein further includes a therapeuticallyeffective amount of an additional therapeutically active agent.

According to still further features in the described preferredembodiments, the additional therapeutically active agent isanti-platelet agent.

According to still further features in the described preferredembodiments, the anti-platelet agent is selected from the groupconsisting of aspirin, clopidogrel, abciximab, argatroban, cilostazol,danaparoid, dazoxiben, dipyridamole, eptifibatide, ticlopidine andtirofiban.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel serotonin reuptakeinhibitors with peripheral-system-restricted activity, as well ascompositions and methods utilizing same, which are far superior to thepresently used SRIs by being substantially devoid of CNS effects.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the term “preventing” includes barring an organism fromacquiring a condition in the first place.

The term “comprising” means that other steps and ingredients that do notaffect the final result can be added. This term encompasses the terms“consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 presents comparative plots showing the inhibition of the bindingof [³H]citalopram (2 nM) to the human platelet membranes serotonintransporter (5-HTT) by N-methyl-citalopram (NMC, marked as “M-cit”) orcitalopram (marked as “cit”) as a function of the concentration of thecompetitive inhibitor, and showing that NMC exhibits a lower bindingaffinity to 5-HTT as that of citalopram (Ki values of 62.00±8.3 nM forNMC and 6.32±1.10 nM for citalopram;

FIG. 2 presents comparative plots showing the inhibition of serotoninuptake in intact freshly prepared human platelets affected by thepresence of the tested compounds, NMC (marked as “M-cit”) and citalopram(marked as “cit”), expressed in percent of inhibition of the uptake of[³H]serotonin uptake (50 nM) as a function of the concentration of thetested compounds, and showing an inhibition constant (Ki) value of4.3±0.9 nM for citalopram and 52.0±15 nM for NMC;

FIG. 3 presents comparative plots showing the inhibition of the bindingof [³H]citalopram binding (1 nM) to rat brain membranes serotonintransporter (5-HTT) by N-methyl-citalopram (NMC; marked as “M-cit”) orcitalopram (marked as “cit”) as a function of the concentration of thecompetitive inhibitor, and showing inhibition constant (Ki) values of53±15 nM for NMC and 3±1 nM for citalopram;

FIG. 4 presents comparative plots showing the inhibition of serotoninuptake in freshly prepared rat brain synaptosomes effected by thepresence of the tested compounds, NMC and citalopram, expressed inpercent of inhibition of [³H]serotonin uptake (50 nM), and showinginhibition constant (Ki) values of 44±6.6 nM for NMC and 4.5±0.7 nM forcitalopram;

FIG. 5 presents comparative plots of the measurement of plateletaggregation as recorded for a sample from a healthy donor, showing thepercent inhibition of human platelet aggregation effected by citalopram(plot denoted “Channel 4” and colored in brown), by N-methyl-citalopram(plot denoted “Channel 3” and colored in blue), and by the control PBS(plot denoted “Channel 2” and colored in red);

FIG. 6 presents a repeat of the experiment presented in FIG. 5 asmeasured for a sample taken from the same healthy donor, showing thepercent inhibition of human platelet aggregation effected by citalopram(plot denoted “Channel 3” and colored in blue), by N-methyl-citalopram(plot denoted “Channel 2” and colored in red), and by the control PBS(plot denoted “Channel 1” and colored in green);

FIGS. 7A-B present plots of [³H]dopamine uptake (FIG. 7A) and[³H]noradrenaline uptake (FIG. 7B) into rat brain synaptosomes as afunction of the concentration of a drug, comparing the inhibitory effectof citalopram, NMC and mazindol; and

FIG. 8 presents comparative plots of the accumulation of[³H]disintegration events per minute (DPM) in the cerebral cortexsamples of mice after the indicated times following intraperitonealinjection of either [³H]N-methyl-citalopram (marked in red circles) or[³H]citalopram (marked in black rectangle) (values represent themean+/−standard deviations for 3 mice for each compound and at each timepoint, with the exception of 40 minutes for which only a single mousewas injected with each compound).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of novel compounds having serotonin reuptakeinhibitory activity, which can be used as drugs in the treatment ofdiseases, disorders and medical conditions wherein regulation ofserotonin levels in the peripheral system is beneficial. Morespecifically, the novel serotonin reuptake inhibitors presented hereinexhibit their activity mainly in the peripheral system while not havinga significant effect on the serotonin levels in the CNS, and cantherefore be used beneficially to treat, for example, chronic diseasesin which elevated platelets aggregation and increasedplatelet-endothelial interactions are part of the disease process, suchas in the case of thrombosis and the related risks of ischemia, ischemicheart disease (IHD), myocardial infarction (MI), cerebral stroke,pulmonary embolism, and type-2 diabetes-associated vascularabnormalities, as well as other diseases and disorders in whichinhibition of peripheral serotonin is beneficial.

As discussed hereinabove, when crossing the blood-brain barrier (BBB),serotonin reuptake inhibitors increase the extracellular level ofserotonin by inhibiting reuptake thereof into the presynaptic cell andthereby increasing the level of serotonin available for binding to thepostsynaptic receptor. In the peripheral system it has been shown thatserotonin reuptake inhibitors reduce the levels of serotonin in theplatelets which serve as storage vessels thereof and hence presentlyknown serotonin reuptake inhibitors, particularly SSRIs were shown tohave anti-platelet effects. Presently known SSRIs, which were developedas antidepressants, are required to penetrate the BBB and exert theireffect on the CNS. However, SSRIs are known to cause several adverseCNS-related effects, such as nausea, drowsiness or somnolence, headache,clenching of teeth, extremely vivid and strange dreams, dizziness,changes in appetite, weight loss/gain, changes in sexual behavior(reduced libido), increased feelings of depression and anxiety, panicattacks, tremors, autonomic dysfunction including orthostatichypotension, increased or reduced sweating, akathisia, depersonalization(derealization), flattened emotions and increased aggressiveness.

Therefore, albeit the amounting evidence that presently known SSRIs arepotent drug candidates for many chronic medical conditions and evenexhibit improved activity compared to other drugs, they are notprescribed for their treatment due to the severe CNS-related adverseeffects.

While conceiving the present invention, the present inventorshypothesized that compounds having serotonin reuptake inhibitory (SRI)activity may be modified so as to reduce their capacity to cross theBBB. It was further hypothesized that presently known selective andnon-selective serotonin reuptake inhibitors, jointly referred to hereinas serotonin reuptake inhibitors (SRIs), modified chemically so as tohave one or more charged groups while not affecting their SRI activity,could be used as drugs for treating medical conditions whereinmodulating the peripheral serotonin level is beneficial. For example,modified SRIs which retain their ability to bind to the peripheral 5-HTTin platelets, would have potential anti-platelet activity similar topresently known SRIs. However, such modified SSRIs would not exhibit thehighly undesired CNS-related adverse effects since charged molecules areknown to lack the capacity to cross the BBB.

The underlying basis for this conception is based on the hypothesis thatSRIs bind to 5-HTT based on molecular recognition factors which are notnecessarily sensitive to changes in the overall charge of the compound.Furthermore, it has been assumed that charged compounds are unable topass the blood-brain barrier (BBB) due to the unique structure of braincapillaries endothelial cells, which include tight junctions instead ofgap junctions between capillary wall cells, thereby prohibiting thepassage of charged molecules. Hence, modifying an SRI so as to have acharged group may have little or no effect on the binding affinity ofthe modified SRI to 5-HTT, but may have a major effect on its capacityto exert SRI activity in the brain.

While reducing the present invention to practice, the present inventorshave demonstrated citalopram, one of the most widely prescribed SSRIs,as an exemplary test compound for such a modification, and preparedN-methyl-citalopram (NMC) which has a positively charged quaternaryammonium group, and a counter anion, marked as A⁻ in the chemicalstructure illustration presented below. This quaternary ammonium group,by not being capable of participating in proton-exchange interactions,is positively charged at a wide range of pH levels, but mostimportantly, this group retains its positive charge at physiological pH.

Citalopram (see chemical structure illustration above) is an SSRIantidepressant drug commonly used to treat depression associated withmood disorders, on occasions in the treatment of body dysmorphicdisorder and anxiety. It's IUPAC name is1-(3-Dimethylamino-propyl)-1-(4-fluoro-phenyl)-1,3-dihydro-isobenzofuran-5-carbonitrile,and it is marketed under the brand-names Celexa™ (U.S., ForestLaboratories, Inc.), Cipramil™, Citrol™, Sipralexa™, Seropram™ (Europeand Australia), Zetalo (India), Celepram™, Clazil™ (Australia), Zentius™(South America, Roemmers) and Cipram™ (Denmark, H. Lundbeck A/S).

Citalopram is typically sold as a racemic mixture, consisting of 50% ofthe R-(−)-citalopram enantiomer and 50% of the S-(+)-citalopramenantiomer. However, it has been found that only the S-(+) enantiomerhas the desired antidepressant effect, and therefore Lundbeck marketsthe S-(+) enantiomer, under the generic name of escitalopram. Whilecitalopram is supplied as a hydrobromide salt, escitalopram is sold asthe oxalate salt, and in both products the salt form allows theseotherwise lipophilic compounds to dissolve in water.

As demonstrated in the Examples section that follows, NMC has beensynthesized and shown to: (a) bind and inhibit the activity of theplatelet serotonin transporter (5-HTT) with similar affinity as itsparent compound citalopram; (b) bind and inhibit the activity of thebrain serotonin transporter with about 10-fold lower affinity comparedwith its parent compound citalopram; and (c) have a substantiallylimited capacity to penetrate the BBB.

Hence, according to one aspect of the present invention, there isprovided a serotonin reuptake inhibitor (SRI) compound, being modifiedso as to include at least one positively charged group which is selectedsuch that the modified SRI compound retains its charge at physiologicalpH, while substantially retaining its SRI activity. Herein andthroughout the SRI compounds presented herein which are being modifiedso as to include at least one positively charged group, are referred tointerchangeably as modified SRIs or modified SRI compounds.

The phrase “serotonin reuptake inhibitor compound”, abbreviated hereinto SRI, as used herein, refers to compounds having the capacity tomodulate the levels of serotonin in the body by inhibiting itsre-absorption by the molecular transporter, 5-HTT. More specifically,SRIs exhibit competitive inhibition of 5-HTT. Serotonin reuptakeinhibitors can be sorted into families according to their specificity,hence selective serotonin reuptake inhibitor (SSRI), includealaproclate, citalopram, escitalopram, etoperidone, fluoxetine,fluvoxamine, paroxetine, sertraline and zimelidine,serotonin-norepinephrine reuptake inhibitor (SNRI), includedesvenlafaxine, duloxetine, milnacipran, nefazodone and venlafaxine, andserotonin-noradrenaline-dopamine reuptake inhibitor (SNDRI), includebrasofensine, tesofensine and nomifensine.

According to preferred embodiments, the serotonin reuptake inhibitor(SRI) compound is a serotonin-selective reuptake inhibitor (SSRI).

The phrase “positively charged group”, as used herein, refers to an atomor a group of atoms which forms a part of an organic molecule, and whichis characterized by a positive electrostatic charge. Compounds whichinclude one or more positively charged groups are molecular ionsoftentimes referred to as molecular cations. A positively charged groupof atoms has at least one electron less than the number of protons inthese atoms. Positively charged groups include, for a non-limitingexample, ammonium and sulfonium groups.

A positively charged group which retains its charge at physiological pHis a group that is not capable of participating in proton-exchangeinteractions at a pH range which is typical to the physiologicalenvironment in the body where the SRI is active. Typically, thephysiological pH is about 7.4; therefore a positively charged groupwhich retains its charge at physiological pH refers to a positivelycharged chemical group that stays ionized in a pH range of about 5-8. Itis noted that even in the GI, where the pH level is extremely low interms of physiological pH, the positively charged group according topreferred embodiments remains positively charged, and hence modified SRIcompounds according to the present invention designed for oraladministration, are not adversely affected by the GI pH levels.

Quaternary ammonium groups are known to be positively charged at any pHrange, including the physiological pH range, hence the positivelycharged group, according to preferred embodiments of the presentinvention, is a quaternary ammonium group.

The phrase “quaternary ammonium”, as used herein, refers to a nitrogenatom which forms a part of a molecule (an amine, as defined hereinbelow)that is attached to four non-hydrogen substituents and thus ispositively charged.

Hence, according to preferred embodiments, the SRI is modified so as tohave a quaternary ammonium group which has the general formula:—(NR₁R₂R₃)⁺Z⁻

wherein:

Z is an organic or inorganic anion; and

R₁, R₂ and R₃ are each independently selected from the group consistingof alkyl, aryl and cycloalkyl.

Preferably, R₁, R₂ and R₃ are each an alkyl having from 1 to 4 carbonatoms, and more preferably, R₁, R₂ and R₃ are each methyl, resulting inthe positively charged group, or the quaternary ammonium group —(NMe₃)⁺.

As used herein, the term “amine” describes a —NR′R″ group where each ofR′ and R″ is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic,aryl or heteroaryl, as these terms are defined herein.

As used herein, the term “alkyl” describes an aliphatic hydrocarbonincluding straight chain and branched chain groups. Preferably, thealkyl group has 1 to 20 carbon atoms, and more preferably 1-10 carbonatoms. Whenever a numerical range; e.g., “1-10”, is stated herein, itimplies that the group, in this case the alkyl group, may contain 1carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including10 carbon atoms. The alkyl can be substituted or unsubstituted. Whensubstituted, the substituent can be, for example, an alkyl, an alkenyl,an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a halide, a hydroxy, analkoxy and a hydroxyalkyl as these terms are defined hereinbelow. Theterm “alkyl”, as used herein, also encompasses saturated or unsaturatedhydrocarbon, hence this term further encompasses alkenyl and alkynyl.

The term “alkenyl” describes an unsaturated alkyl, as defined herein,having at least two carbon atoms and at least one carbon-carbon doublebond. The alkenyl may be substituted or unsubstituted by one or moresubstituents, as described hereinabove.

The term “alkynyl”, as defined herein, is an unsaturated alkyl having atleast two carbon atoms and at least one carbon-carbon triple bond. Thealkynyl may be substituted or unsubstituted by one or more substituents,as described hereinabove.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. The aryl groupmay be substituted or unsubstituted by one or more substituents, asdescribed hereinabove.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted by one or more substituents, as describedhereinabove. Representative examples are thiadiazole, pyridine, pyrrole,oxazole, indole, purine and the like.

The positively charged group can be formed on the SRI from an existinggroup which forms a part of the SRI, namely, by turning a partiallycharged or uncharged group into a positively charged group, or byturning an existing positively charged group which can participate inproton-exchange interaction into one that cannot participate in suchinteraction, making it into an irreversible positive charge, or apermanent positive charge, thereby modifying the SRI.

Alternatively, the positively charged group can be added to the SRI bysubstituting one or more carbon atom with a positively charged group,e.g., by replacing a hydrogen atom or any other substituent with aquaternary ammonium or a tertiary sulfonium group.

The present embodiments further encompass any enantiomers, prodrugs,solvates, hydrates and/or pharmaceutically acceptable salts of thecompounds described herein.

As used herein, the term “enantiomer” refers to a stereoisomer of acompound that is superposable with respect to its counterpart only by acomplete inversion/reflection (mirror image) of each other. Enantiomersare said to have “handedness” since they refer to each other like theright and left hand. Enantiomers have identical chemical and physicalproperties except when present in an environment which by itself hashandedness, such as all living systems.

The term “prodrug” refers to an agent, which is converted into theactive compound (the active parent drug) in vivo. Prodrugs are typicallyuseful for facilitating the administration of the parent drug. They may,for instance, be bioavailable by oral administration whereas the parentdrug is not. A prodrug may also have improved solubility as comparedwith the parent drug in pharmaceutical compositions. Prodrugs are alsooften used to achieve a sustained release of the active compound invivo. An example, without limitation, of a prodrug would be a compoundof the present invention, having one or more carboxylic acid moieties,which is administered as an ester (the “prodrug”). Such a prodrug ishydrolyzed in vivo, to thereby provide the free compound (the parentdrug). The selected ester may affect both the solubility characteristicsand the hydrolysis rate of the prodrug.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the compound of the present invention) and a solvent, wherebythe solvent does not interfere with the biological activity of thesolute. Suitable solvents include, for example, ethanol, acetic acid andthe like.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. Herein, the phrases“physiologically suitable” and “pharmaceutically acceptable” areinterchangeably used and refer to an approved substance that does notcause significant irritation to an organism and does not abrogate thebiological activity and properties of the administered agent.

Since the modified SRI according to the present embodiments is a chargedcompound by definition, namely it is in a cation form, the phrase“pharmaceutically acceptable salt” refers to other charged species ofthe SRI compound, i.e. zwitterion or multiple charged species thereofand its counter ion(s), which is typically used to modify the solubilitycharacteristics of the parent compound and/or to reduce any significantirritation to an organism by the parent compound, while not abrogatingthe biological activity and properties of the administered compound.Non-limiting examples for an anion include chloride, bromide, oxalate,maleate, mesylate and the like.

According to further preferred embodiments, the SRI is modified so as tohave a sulfonium group which has the general formula:—(SR₄R₅)⁺Z⁻

wherein:

Z is an organic or inorganic anion; and

R₄ and R₅ are each independently selected from the group consisting ofalkyl, aryl and cycloalkyl.

R₄, and R₅ are preferably each an alkyl having from 1 to 4 carbon atoms,and more preferably, R₄ and R₅ are each methyl, resulting in thepositively charged group, or the sulfonium —(SMe₂)⁺.

The term “sulfonium”, as used herein, refers to a —S⁺R′R″, wherein R′and R″ are each independently alkyl, cycloalkyl, heteroalicyclic, arylor heteroaryl.

Exemplary SRIs from which the compounds described herein can be derivedinclude, without limitation, SSRIs such as citalopram, alaproclate,dapoxetine, etoperidone, fluoxetine, fluvoxamine, paroxetine,sertraline, venlafaxine and zimelidine. These compounds are presented inTable 1 below.

Other SRIs which inhibit the reuptake of serotonin, including, forexample, SNRIs and SNDRIs such as, for example, desvenlafaxine,duloxetine, milnacipran, nefazodone, venlafaxine, brasofensine,tesofensine and nomifensine, may also be modified so as to have apositively charged group, and be used in a similar way as the SSRIspresented herein.

Table 1 presents some of the presently known SSRI including their tradenames and chemical structure.

TABLE 1 SSRI Name Trade name(s) Structure Alaproclate N/A

Etoperidone N/A

Citalopram Celexa, Cipramil, Seropram, Celepram, Ciazil, Emocal, Sepram,Seropram

Escitalopram Lexapro, Cipralex, Esteria

Fluoxetine Prozac, Fontex, Seromex, Seronil, Sarafem, Fluctin

Fluvoxamine Luvox, Faverin, Fevarin Dumyrox

Paroxetine Paxil, Seroxal, Aropax, Deroxat, Rexetin, Xetanor, Paroxat

Sertraline Zoloft, Sertralin, Lustral, Apo-Sertral, Asentra, Gladem,Serlift, Stimuloton, Xydep, Serlain, Concorz

Dapoxetine N/A

Venlafaxine Depurol, Dobupal, Efectin, Efexor, Elafax, Faxine, Flavix,Norpilen, Trevilor, Vandral, Velafax, Venlafaxina, Venlax, Venlor,Viepax

Zimelidine Normud, Zelmid

As can be seen in Table 1, all these SSRIs comprise at least one aminegroup, which can be readily converted into a quaternary ammonium, i.e. apositively charged group. As can further be seen in Table 1, all theseSSRIs offer a multitude of substitution positions on which a positivelycharged group can be added. One of the requirements for these chemicalmodifications is the retention of the SSRI activity.

For example, etoperidone can be methylated at one or both of thepiperazine nitrogen atoms which will turn each of these amines into aquaternary ammonium. Fluvoxamine can be modified so as to have one ofthe aliphatic carbon atoms substituted with a thiol group, which can befurther modified into a tertiary sulfonium group.

As mentioned hereinabove, the present inventors selected citalopram as amodel to demonstrate the present embodiments, and preparedN-methyl-citalopram (NMC), as exemplified in the Examples section thatfollows. As further demonstrated in the Examples section that follow,NMC was found to be a promising drug candidate for a peripherally-actingSSRI drug lacking CNS activity.

As is demonstrated in the Examples section that follows, NMC recognizedthe human platelet 5-HTT with lower affinity as compared to citalopram,shown by competition binding experiments in human platelet membranes,using [³H]citalopram as a detectible marker. NMC was also shown toinhibit [³H]5-HT uptake by freshly isolated human platelets, yet in ahigher inhibition constant (Ki) value than determined for citalopram.

NMC was about one-order of magnitude less potent than citalopram inbinding to and inhibiting 5-HTT in rat brain.

NMC appears to penetrate the BBB to a much lower extent than citalopram,at levels that were near the detection threshold in the performedexperiments. It is estimated that the brain penetration ofN-methyl-citalopram following its intraperitoneal injection is about50-fold less than that of citalopram.

Citalopram and NMC both exhibited strong inhibition of human plateletaggregation.

These data therefore demonstrate that NMC is a potent anti-plateletdrug, whereby being a quaternary nitrogen (quaternaryammonium)-containing compound (positively charged at any physiologicalpH) renders its capability to cross the BBB substantially limited.Therefore, NMC and other modified (e.g., quaternary ammonium-containing)SRI compounds are potent anti-platelet agents (decreasing plateletaggregation) which are devoid of the undesired CNS effects of presentlyknown SRIs.

According to another aspect of the present invention, there is provideda process of preparing the SRI presented herein, the process is effectedby modifying an SRI so as to include one or more positively chargedgroup which retains its charge at physiological pH, while substantiallyretaining its SRI activity.

Preferably, the process is effected by alkylating or arylating anexisting chemical group on the SRI, such as an amine group.Alternatively, an amine group and/or a sulfonium group can be added tothe SRI.

When alkylating or arylating a pre-existing amine group, the process iseffected by N-alkylating or N-arylating this amine group.

N-Alkylation and N-arylation are typically effected by utilizing analkylating/arylating agent. The phrase “alkylating/arylating agent”, asused herein, refers to a chemical reagent which use thereof can place analkyl or an aryl, as this is define herein, at a designated position ona given reactant compound.

Exemplary alkylating or arylating agents include, without limitation, analkyl- or arylsulfonate, an alkyleneimine, phosgene, an alkyl- oraryl-tosylates, an alkyl- or aryl-triflates, an alkyl- or aryl-halide, adialkyl- or diaryl-sulfate, an alumoxanes, a trialkylaluminum and atris(trialkylyl)aluminum. Preferably the alkylating agent is methyliodide.

Alkylation (methylation) reactions are commonly used and practiced onamines and other chemical groups for many decades, and may involve otherpreparative steps such as conversion of a charged amine in a watersoluble parent compound into the organic-solvent soluble free-base formthereof, typically by means of a bi-phasic reaction with a mild base,such as sodium bicarbonate, which allows the free-base form of theparent compound to cross to the organic phase once losing its charge.

The phrase “free-base form”, as used herein, refers to a standalonebasic form of a non-quaternary amine group, as opposed to itswater-soluble ammonium salt form. For example R—NH₂ is the free-baseform of R—NH₃ ⁺.

Following similar synthetic paths, additional novel SRIs having apositively charged group can be obtained by a single, double or tripleN-methylation of an amine group which already form a part of themolecule, including, for example, N,N,N-trimethyl-alaproclate,N-methyl-dapoxetine, N-methylpiperazin-etoperidone,N,N-dimethyl-fluoxetine, N,N,N-trimethyl-fluvoxamine,N,N-dimethylpiperidine-paroxetine, N,N-dimethyl-sertraline andN-methyl-venlafaxine.

As discussed hereinabove and demonstrated in the Examples section thatfollows, an SRI modified to have at least one positively charged groupis a very promising drug candidate that can be beneficially used totreat medical conditions which are associated with peripheral serotoninlevels.

According to another aspect of the present invention, there is provideda method of modulating a level and/or activity of peripheral serotonin(5-HT) in a subject while substantially maintaining a level and/oractivity of central serotonin in the subject, the method is effected byadministering to the subject an SRI modified so as to comprise at leastone positively charged group which retains its charge at physiologicalpH, while substantially retaining its SRI activity.

Accordingly, there is provided a use of the modified SRI as presentedherein in the modulation of a level and/or activity of peripheralserotonin (5-HT) in a subject while substantially maintaining a leveland/or activity of central serotonin in the subject.

The phrase “modulating the level and/or activity” as used herein refersto the result of an intended action which indirectly alters (increase ordecrease) the concentration (level), and/or the activity of a substanceat certain intended bodily sites in a subject.

The phrase “substantially maintaining a level and/or activity” as usedherein, means the opposite of the previous definition, namely, theconcentration (level), and/or the activity of a substance at certainintended bodily sites in a subject does not alter (increase or decrease)as a result of an intended action.

In the context of the present embodiments, the modified SRI presentedherein have the ability to modulate the level and/or activity ofserotonin in the peripheral system while at the same time not affecting(substantially maintaining) the level and/or activity of serotonin inthe CNS.

According to preferred embodiments, this method of modulating a leveland/or activity of peripheral serotonin can be used for treating certainmedical conditions in which this type of modulating is beneficial. Suchmedical conditions include, without limitation, cardiovascular diseasesor disorders, cerebrovascular diseases or disorders, ischemic heartdisease (IHD), myocardial infarction (MI), cerebral stroke, pulmonaryembolism, type-2 diabetes-associated vascular abnormalities, pulmonaryarterial hypertension, peripheral arterial occlusive disease, rheumatoidarthritis, autoimmune disorders, kidney failure, inflammatory boweldisease, acute coronary syndrome, coronary artery disease (CAD) andrestenosis following coronary artery bypass post-grafting.

As discussed hereinabove, peripheral serotonin levels have beenimplicated mainly with platelet aggregation, and in turn, plateletaggregation has been implemented with several medical conditions, mostof which are highly lethal. These include cardiovascular diseases ordisorders, IHD, MI, stroke, pulmonary embolism, diabetes-associatedvascular abnormalities, pulmonary hypertension and arterial occlusivediseases, as well as preventive measures against clot formation andother prophylactic anti-embolic treatments.

Hence, according to yet another aspect of the present invention, thereis provided a method of treating a disease or disorder in which reducingor preventing platelet aggregation and/or platelet-endothelialinteractions is beneficial, the method is effected by administering to asubject in need thereof a therapeutically effective amount of the SRImodified so as to comprise at least one positively charged group whichretains its charge at physiological pH, while substantially retainingits SRI activity.

The term “therapeutically effective amount” or “pharmaceuticallyeffective amount” denotes that dose of an active ingredient or acomposition comprising the active ingredient that will provide thetherapeutic effect for which the active ingredient is indicated.

Accordingly, there is provided a use of the modified SRI as presentedherein in the treatment of a disease or disorder in which reducing orpreventing platelet aggregation and/or platelet-endothelial interactionsis beneficial.

In all the methods and uses presented herein, the modified SRI issubstantially incapable of modulating the serotonin level in the CNS ofa subject, may it be increasing or decreasing CNS serotonin levels,thereby avoiding CNS-related effects.

The SRIs of the present invention when used, for example, as preventivemeasures against clot formation or a prophylactic anti-embolictreatment, can be utilized in combination with an additional therapeuticagent, preferably an anti-platelet agent and other inhibitors ofplatelet function.

Examples of useful inhibitors of platelet function include, but are notlimited to acadesine, anagrelide (given at doses exceeding 10 mg/day),anipamil, argatroban, aspirin, clopidogrel, cyclooxygenase inhibitorssuch as nonsteroidal anti-inflammatory drugs and the synthetic compoundFR-122047, clofibrate, caffeine, danaparoid sodium, dazoxibenhydrochloride, diadenosine 5′,5″-P1,P4-tetraphosphate (Ap4A) analogs,difibrotide, dilazep dihydrochloride, 1,2- and 1,3-glyceryl dinitrate,dipyridamole, dopamine and 3-methoxytyramine, efegatran sulfate,enoxaparin sodium, glucagon, glycoprotein IIb/IIIa antagonists such asRo-43-8857 and L-700,462, ifetroban, ifetroban sodium, iloprost,isocarbacyclin methyl ester, isosorbide-5-mononitrate, itazigrel,ketanserin and BM-13.177, lamifiban, lifarizine, molsidomine,nifedipine, oxagrelate, PGE, platelet activating factor antagonists suchas lexipafant, prostacyclin (PGI₂), pyrazines, pyridinol carbamate,ReoPro (i.e., abciximab), sulfinpyrazone, synthetic compounds BN-50727,BN-52021, CV-4151, E-5510, FK-409, GU-7, KB-2796, KBT-3022, KC-404,KF-4939, OP-41483, TRK-100, TA-3090, TFC-612 and ZK-36374,2,4,5,7-tetrathiaoctane, 2,4,5,7-tetrathiaoctane 2,2-dioxide,2,4,5-trithiahexane, theophyllin pentoxifyllin, thromboxane andthromboxane synthetase inhibitors such as picotamide and sulotroban,ticlopidine, tirofiban, trapidil and ticlopidine, trifenagrel,trilinolein, 3-substituted 5,6-bis(4-methoxyphenyl)-1,2,4-triazines, andantibodies to glycoprotein IIb/IIIa as well as those disclosed in U.S.Pat. No. 5,440,020, and any other anti-serotonin drug.

More preferably, the anti-platelet agent is aspirin, clopidogrel,abciximab, argatroban, cilostazol, danaparoid, dazoxiben, dipyridamole,eptifibatide, ticlopidine or tirofiban.

Other therapeutic agents which may be used beneficially to treat medicalconditions which are associated with platelet aggregation includeanticoagulants such as heparin, warfarin, acenocoumarol, phenprocoumon,phenindione and hirudin, as well as nitric oxide (NO) donating agents.

The modified SRIs presented herein can be utilized per-se or as a partof a pharmaceutical composition, hence, according to still anotheraspect of the present invention, there is provided a pharmaceuticalcomposition which include, as an active ingredient, the SRI presentedherein and a pharmaceutically acceptable carrier.

The pharmaceutical compositions or medicaments presented herein mayinclude additional therapeutic agents, as these are discussedhereinabove.

As used herein a “pharmaceutical composition” refers to a preparation ofthe SRI presented herein, with other chemical components such aspharmaceutically acceptable and suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered SRI. Examples, without limitations, of carriers are:propylene glycol, saline, emulsions and mixtures of organic solventswith water, as well as solid (e.g., powdered) and gaseous carriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of adrug. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the SRIs into preparationswhich can be used pharmaceutically. Proper formulation is dependent uponthe route of administration chosen. The dosage may vary depending uponthe dosage form employed and the route of administration utilized. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p. 1).

The pharmaceutical composition may be formulated for administration ineither one or more of routes depending on whether local or systemictreatment or administration is of choice, and on the area to be treated.Administration may be done orally, by inhalation, or parenterally, forexample by intravenous drip or intraperitoneal, subcutaneous,intramuscular or intravenous injection, or topically (includingophtalmically, vaginally, rectally, intranasally).

Formulations for topical administration may include but are not limitedto lotions, ointments, gels, creams, suppositories, drops, liquids,sprays and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets, pills,caplets, capsules or tablets. Thickeners, diluents, flavorings,dispersing aids, emulsifiers or binders may be desirable.

Formulations for parenteral administration may include, but are notlimited to, sterile solutions which may also contain buffers, diluentsand other suitable additives. Slow release compositions are envisagedfor treatment.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising an SRI of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof a particular medical condition, disease or disorder, as is detailedhereinabove.

According to preferred embodiments, the pharmaceutical composition isbeing packaged in a packaging material and identified in print, in or onthe packaging material for use in the treatment of a medical conditionin which modulating a level and/or activity of peripheral serotonin(5-HT) in a subject, while substantially maintaining a level and/oractivity of central serotonin, is beneficial, as these are discussedhereinabove.

According to other preferred embodiments, the pharmaceutical compositionis being packaged in a packaging material and identified in print, in oron the packaging material for use in the treatment of a disease ordisorder in which reducing or preventing platelet aggregation and/orplatelet-endothelial interactions is beneficial, as these are discussedhereinabove.

Accordingly, there is provided a use of the modified SRI as presentedherein in the manufacture of a medicament for treating a disease ordisorder in which reducing or preventing platelet aggregation and/orplatelet-endothelial interactions is beneficial.

Alternatively, there is provided a use of the modified SRI as presentedherein in the manufacture of a medicament for treating a medicalcondition in which modulating a level and/or activity of peripheralserotonin (5-HT) in a subject while substantially maintaining a leveland/or activity of central serotonin is beneficial.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Materials and Experimental Methods

Citalopram, serotonin (5-HT), fluoxetine, mazindol, dopamine andnoradrenaline of the highest obtainable purity were obtained fromSigma-Aldrich Israel Ltd.

[³H]Serotonin ([³H]5-HT, 20.3 Ci/mmol), [³H]Citalopram (79 Ci/mmol),[³H]Dopamine (55.1 Ci/mmol) and [³H]Ketanserin (76 Ci/mmol), wereobtained from Perkin Elmer Life Sciences (Boston, Mass., USA).

[³H]Noradrenaline (35 Ci/mmol) was purchased from Amersham, GEHealthcare UK.

Homogenization of samples was conducted using a Kinematica Polytron®Homogenizer device, Westbury, N.Y., USA.

Brain tissue samples were grinded using a Potter-Elvehjem, PTFE pestleand glass tube grinder.

Tritium concentration, expressed in DPM, was determined using aβ-emission liquid scintillation analyzer, Tri-Carb 2100TR by Packard.

Male Wistar rats and Imprinting Control Region (ICR) mice (Harlanlaboratories, Israel) were maintained under controlled light andtemperature conditions, with food and water provided ad libitum.

Animal procedures were approved by the Animal Care Committees ofTel-Aviv University (Approval numbers: M-06-105, M-07-027).

The independent samples t-test, using SPSS software (SPSS Inc. Chicago,Ill.), was used as appropriate. Results are expressed as means±S.E.M.

Preparation of N-methyl-citalopram:

N-methyl-citalopram (NMC, 1.8 grams) was synthesized from of thefree-base form of the parent compound citalopram via methylation bymethyl iodide. Saturated solution of sodium bicarbonate (20 ml) wasadded to a solution of citalopram (1 gram) in dichloromethane (20 ml).The reaction mixture was mixed for a few minutes, and then was allowedto separate to two phases. The organic phase was concentrated underreduced pressure to give 0.5 grams of the free-base form of citalopramthat was used in the next step without further purification.

Methyl Iodide (10 equivalents) was added to a cool solution thefree-base form of citalopram (1 gram, 3.08 mmol) in acetone. The mixturewas heated to reflux over night, and thereafter the solvent was removedunder reduced pressure to produce N-methyl citalopram (NMC, 0.5 grams,1.5 mmol, 50 5 yield). The purity of NMC and the absence of citalopramwere verified by HPLC.

Preparation of [³H]N-methyl-citalopram:

[³H]Methyl iodide (C[³H]₃I), at specific activity of 70 Ci/mmol, wasvacuum transferred into a tritiation production vessel containing thefree base form of citalopram. Following several hours of incubation allvolatiles were removed via distillation under reduced pressure. Thecrude residue was purified by HPLC to yield [³H]N-methyl-citalopram, atmore than 99% radiochemical purity.

Preparation of Human Platelet Membrane for Binding Assays:

Human blood samples (25 ml) were collected in the morning (between 8 and10 AM) from healthy volunteers into plastic tubes containing 1 mM EDTAas anticoagulant. Platelet-rich plasma was separated from blood cells bylow-speed centrifugation (350 g for 10 minutes), diluted in 20 ml of 50mM Tris/HCl buffer pH 7.4 (containing 120 mM NaCl and 5 mM KCl) andcentrifuged at 1700 g for 20 minutes. The supernatant was discarded andthe final membrane pellet was kept frozen at −70° C. until used, usuallywithin 2 days.

On the day of use, the platelet membrane pellet was disrupted withBrinkman polytron in 20 ml of 50 mM Tris HCl buffer pH 7.4 (containing120 mM NaCl and 5 mM KCl) and centrifuged twice at 27,000 g for 20minutes. It was then resuspended in 9 ml buffer to yield a final proteinconcentration of about 0.8 mg/ml.

Assay of [³H]Citalopram Binding to Human Platelets:

[³H]Citalopram binding to 5-HT transported (5-HTT) was determined usinga method modified from Plenge et al. [20]. A standard binding assaycontained: 200 of homogenate, 100 μl [³H]citalopram (2 nM) and 50 μlbuffer or test drug. After a 60 minutes incubation period at 25° C.,homogenates were diluted in 3 ml ice-cold buffer and filtered throughWhatman GF/C glass fiber filters. Filters were washed three times with 3ml ice-cold buffer, and the radioactivity was measured in scintillationliquid in a β-counter. Specific binding was defined as the differencebetween total [³H]citalopram binding (triplicate samples) and thebinding in the presence of 10 μM fluoxetine (duplicate samples).

Assay of [³H]Citalopram Binding to Rat Brain Membranes:

Rat brain cortex was disrupted with Brinkman polytron in 50 volumes of50 mM Tris HCl buffer, pH 7.4 (containing 120 mM NaCl and 5 mM KCl) andcentrifuged (three times) at 30,000 g for 10 minutes. It was thenresuspended in the same buffer to yield a final concentration of about21 mg/ml (wet weight). [³H]Citalopram binding was determined using amethod modified from Plenge et al. [20]. A standard binding assaycontained: 100 μl of homogenate, 100 [³H]citalopram (1 nM) and 300 μlbuffer or 250 μl buffer and 50 μl test drug. After a 60-minutesincubation period at 25° C., homogenates were diluted in 3 ml ice-coldbuffer and filtered through Whatman GF/C glass fiber filters. Filterswere washed three times with 3 ml ice-cold buffer, and the radioactivitywas measured in scintillation liquid in a β-counter. Specific bindingwas defined as the difference between total [³H]citalopram binding(triplicate samples) and the binding in the presence of 10 μMfluvoxamine (duplicate samples).

Isolation of Human Platelets for Measuring [³H]5-HT Uptake:

Human blood samples (25 ml) were collected in the morning (between 8 and10 AM) from healthy volunteers into plastic tubes containing 1 mM EDTAas anticoagulant. Platelet-rich plasma (PRP) was separated by low speedcentrifugation (350 g for 10 minutes). The PRP was diluted 1:1 in bufferA (19 mM phosphate buffer, 0.119 mM NaCl, 3.9 mM KCl, 0.65 mM MgSO₄,0.51 mM CaCl₂, 2 mg/ml glucose, 0.2 mg/ml ascorbic acid, 1.6×10⁻⁴ M EDTAand 5.0×10⁻⁵ M pargyline, at pH 7.4) and centrifuged at 1700 g for 20minutes. The platelet pellet was gently resuspended in buffer A (10⁸-10⁹platelets per ml) and used to measure [³H]5-HT uptake.

Assay of [³H]5-HT Uptake by Human Platelets:

[³H]5-HT uptake assays were carried out as described previously [21]. Astandard assay contained 200 μl washed platelets, 50 μl [³H]5-HT and 50μl buffer A or test drug. The tubes were preincubated at 37° C. for 10minutes, at which time [³H]5-HT was added (50 nM). After a 2 minuteincubation period at 37° C. the reaction was stopped by rapidly coolingthe tubes on ice and the mixture was filtered under vacuum on glassfiber filters (GF/C). The filters were washed with ice cold buffer A andthe radioactivity was counted in scintillation liquid in a β-counter.Specific uptake was defined as the difference between total [³H]5-HTuptake at 37° C. (triplicate samples) and the uptake measured at 0° C.(duplicate samples).

Measurement of [³H]5-HT Uptake to Rat Brain Synaptosomes:

Rat brain cortex sample was homogenized in 10 volumes of 0.32 M sucroseusing a Teflon-glass grinder. The homogenate sample was centrifuged at1000 g for 10 minutes and the resulting supernatant (S1) containingsynaptosomes was used to measure [³H]5-HT uptake.

[³H]5-HT uptake assays were carried out as described previously [21]. Astandard assay contained 50 μl synaptosomes, 50 μl [³H]5-HT and 900 μlbuffer A (19 mM phosphate buffer, 0.119 mM NaCl, 3.9 mM KCl, 0.65 mMMgSO₄, 0.51 mM CaCl₂, 2 mg/ml glucose, 0.2 mg/ml ascorbic acid, 1.6×10⁻⁴M EDTA and 5.0×10⁻⁵ M pargyline, at pH 7.4) or 850 μl buffer A and 50 μltest drug. The tubes were pre-incubated at 37° C. for 10 minutes, atwhich time [³H]5-HT (50 nM) was added. After a 4 minute incubationperiod at 37° C. the reaction was stopped by rapidly cooling the tubeson ice and the mixture was filtered under vacuum on glass fiber filters(GF/C). The filters were washed with ice cold buffer A and theradioactivity was counted in scintillation liquid in a β-counter.Specific uptake was defined as the difference between total [³H]5-HTuptake at 37° C. (triplicate samples) and the uptake measured at 0° C.(duplicate samples).

[³H]Ketanserin Binding to Rat Brain Membranes:

[³H]ketanserin binding experiment was performed according to Takao etal. [Eur. J. Pharmcol., 1997, 333, p. 123-8]. Briefly, frozen rat braincortex was homogenized in 10 volumes of ice cold 0.32 M sucrose using aTeflon-glass homogenizer. The homogenate was centrifuged at 1000 g for10 minutes at 4° C. and the resulting supernatant was centrifuged at34000 g for 20 minutes at 4° C. The obtained pellet was homogenized withpolytron in 40 volumes 50 mM Tris-HCl buffer pH 7.4 before incubated for15 min at 37° C. The homogenate was finally centrifuged at 38000 g for20 min and pellet resuspended in buffer (10 mg protein/ml) and kept at−70° C. until further use.

A standard binding assay contained 200 μl of brain membranes, 100 μl[³H]ketanserin (0.4 nM) and 700 μl buffer or 600 μl buffer and 100 μltest drug. [³H]ketanserin was added to the test tubes after a 3-minpreincubation period at 37° C. preceding the 15-minute incubation at 37°C. The homogenates were diluted in 3 ml ice-cold buffer and filteredthrough Whatman GF/C glass fiber filters pre-incubated in 0.05%polyethyleneimine. Specific binding was defined as the differencebetween total [³H]ketanserin binding (triplicate samples) and thebinding in the presence of 10 μM mianserin (duplicate).

Preparation of Human Platelet Samples for Platelet AggregationMeasurements:

One of the key and essential characteristics of the compounds presentedherein is related to inhibition of platelet aggregation by the compoundswhich do not penetrate the brain and are therefore devoid of CNSeffects. It is expected that the potent anti-platelet effects ofN-methyl-citalopram would be achieved by in-vivo administration.Presented below is the in-vitro assay, measuring the activity ofN-methyl-citalopram as a human platelet aggregation inhibitor afteraggregation was induced using serotonin (5-HT).

Fresh human platelet-rich plasma (PRP) was prepared as follows: humanplatelets were tested for aggregation within 2 hours of withdrawing 10ml venous blood samples from healthy volunteers, aged between 25-35years and not taking any drug. Venous blood was withdrawn into citrateVacutainer tubes (BD #9NC 0.105M) using Vacuette Holdex from GreinerBio-one (catalogue number 450263) and winged blood collection set(Greiner bio-one #450153). Tubes were mixed gently by inverting them afew times immediately following blood withdrawal. Platelet-rich plasma(PRP) was prepared 45 minutes after blood collection by centrifugationfor 10 minutes at 110 g at room temperature in a swinging-bucketcentrifuge. The PRP samples obtained were transferred into new tubes andthe bloods were centrifuged again at 1900 g for 10 min to yieldplatelet-poor plasma (PPP). For each PRP sample, the aggregometer wascalibrated with the corresponding PPP sample from the same donor.

Measurements of ADP Induced Human Platelet Aggregation:

Platelet aggregation was measured in PRP using an Helena PACKS4aggregometer (Helena Laboratories, Beaumont, Tex.), following a methoddescribed previously [22]. Samples consisting of 198 μl freshly preparedhuman PRP and 22 μl tested compounds were incubated at 37° C. for 15minutes in the aggregometer. Stock solutions of citalopram orN-methyl-citalopram were dissolved in calcium-free phosphate-bufferedsaline (PBS; Biological Industries, Israel, Cat. 02-023-5A) to yieldconcentrations of 10-fold the final indicated concentration. For controlmeasurements 22 μl of PBS were added.

At the last 30 seconds of incubation 11 μl serotonin were added (finalconcentration 50 μM serotonin; freshly prepared serotonin solution inPBS) followed 30 seconds later by 25 μl adenosine diphosphate (ADP;final concentration 1.25 μM; freshly prepared ADP solution in PBS) atthe end of the incubation. Measurements for platelet aggregation in theaggregometer continued for 5 minutes from the time of ADP addition (timepoint referred to as “zero”).

Determination of [³H]N-Methyl-Citalopram BBB Penetration In-Vivo:

These in-vivo experiments were performed for demonstrating that[³H]N-methyl-citalopram does not penetrate the mouse brain followingintraperitoneal injections.

[³H]N-methyl-citalopram was prepared by Vitrax (CA, USA) to a specificradioactivity of 70 Ci/mmol and shown by HPLC to be chemically identicalto non-labeled methyl-citalopram. In comparison, [³H]citalopram had aspecific radioactivity of 79 Ci/mmol.

Each of the radioactive compounds, [³H]N-methyl-citalopram and [³H]citalopram, was diluted with the same non-radioactive compound so thatthe dose of the injected compound was 1.5 μg per gram of body weight(weight range 31-43 grams; injection volume range 155-215 μl in saline).The amount of radioactivity (in injected disintegrations per minute,DPM) of the [³H]-labeled compound was 318,000 DPM per gram weight of[³H]N-methyl-citalopram or 174,000 DPM per gram weight of[³H]citalopram. This protocol allowed the amount of injected citalopramdose to be similar to the higher end of the human clinical recommendeddosage of 1.5 mg citalopram per kg body weight.

Mice were given 20 mg/kg pentobarbital intraperitoneal injection 10minutes prior to being perfused with saline for about 2 minutes. Theperfusion is required for removing residual blood from the brain tissuesand the use of pentobarbital is obligatory due to animal welfareconsiderations. Saline/heparin perfusion was started at 5, 10, 20 or 40minutes from the time of intraperitoneal injections of the radioactivecompounds. At the end of the perfusion, mice heads were cut off andtheir dissected brains were washed with ice-cold saline for 30 seconds.The cerebral cortex tissues were dissected according to the Atlas of theMouse Brain [23] and immediately weighed (weight range: 132-205 mg). Thetissues were thereafter homogenized in 10 volumes of distilled waterusing a Teflon/glass grinder. The radioactivity, expressed as DPM in 500μl homogenate samples, was counted in a liquid scintillation counter(model 2100TR, Packard, USA) and corrected for DPM using an internalstandard.

[³H]Dopamine and [³H]Noradrenaline Uptake by Rat Brain Synaptosomes:

[³H]dopamine and [³H]noradrenaline uptake assays were carried out insynaptosomes from rat brain cortex. After a 10 minutes pre-incubationperiod at 37° C., [³H]dopamine or [³H]noradrenaline were added to thetubes containing synaptosomes and tested drugs. The reactions werestopped after four minutes by rapid cooling on ice. Specific uptake wasdefined as the difference between total uptake at 37° C. (triplicatesamples) and the uptake measured at 0° C. (duplicate samples). Threeseparate experiments were performed.

Determination of Bleeding Time in SSRI and Aspirin Treated Mice:

The anti-thrombotic effects of a drug can be evaluated in mice through ableeding time experiment.

Mice were treated by the tested drugs for two weeks, then anesthetizedand placed on a warming pad before a segment of their tail wasamputated. The tail was immersed in 0.9% isotonic saline at 37° C., andthe time required for the stream of blood to stop was defined as thebleeding time.

Different cuts and drug doses were tested. Control mice wereadministered either DDW (p.o) or saline (i.p). In some experiments (Nos.5 and 6), half of the mice were administered DDW and half saline.

Effect of SSRIs in Induced Pulmonary Thromboembolism in Mice:

An experimental model of acute pulmonary thromboembolism was induced inmice by injecting a mixture of collagen (0.5 mg/kg) and epinephrine (0.5mg/kg). Mice treated chronically with aspirin (70 mg/kg), citalopram (10mg/kg), NMC (10 mg/kg) or saline for two weeks were injected via veintail with collagen-epinephrine solution. The injection was followed by amassive activation of circulating platelets and the widespread formationof platelet thrombi in the microcirculation of the lungs leading todisseminated pulmonary microembolism and paralysis of the animal.

Toxicology Preliminary Experiment:

ICR mice were administered (i.p.) once a day high doses of citalopram orNMC (2 or 3 mice per dose). These mice were then observed for 1 hour.The doses were up to 10-fold higher than the highest doses used in thebleeding time or the pulmonary thromboembolism experiments presentedhereinabove.

EXPERIMENTAL RESULTS In Vitro Studies

Preparation of N-methyl-citalopram:

N-methyl-citalopram (NMC, 1.8 grams) was synthesized via methylation ofcitalopram, using methyl iodide. The purity of NMC and the absence ofcitalopram were verified by HPLC.

Assay of [³H]Citalopram Binding to Human Platelets:

The affinity of N-methyl-citalopram (NMC) to the 5-HT transported(5-HTT) in human platelets was compared to the affinity of citalopram,as presented hereinabove.

FIG. 1 presents comparative plots, showing the inhibition of the bindingof [³H]citalopram to the human platelet membranes serotonin transporterby NMC or citalopram as a function of the concentration of thecompetitive inhibitor. The assay was carried out by competition bindingexperiments carried out in human platelet membranes, using[³H]citalopram as the labeled ligand.

Table 2 present the [³H]citalopram binding and [³H]5-HT uptakeinhibition constants in rat brain and human platelets. In Table 2,values represent the means±S.E.M. obtained from 2-3 independentexperiments each conducted in triplicate. See Methods for furtherdetails.

TABLE 2 Ki (nM) Ki (nM) rat brain human platelets [³H]citalopram[³H]5-HT [³H]citalopram [³H]5-HT binding uptake binding uptakeCitalopram  3 ± 1 4.5 ± 0.7 6.32 ± 1.10  4.3 ± 0.9 NMC 53 ± 15^(a)  44 ±6.6^(b)   62 ± 8.3^(c) 52.0 ± 15^(d) A significant difference of Kivalues was observed in rat brain: ^(a)p = 0.029, ^(b)p = 0.001 versuscitalopram as well as in platelets ^(c)p < 0.0001, ^(d)p = 0.193 (n = 2)versus citalopram.

As can be seen in FIG. 1 and Table 2, N-methyl-citalopram recognized thehuman platelet 5-HTT with lower (about 10-fold) affinity as compared tothat of to citalopram (Ki values of 62.00±8.3 nM and 6.32±1.10 nM forNMC and citalopram, respectively).

FIG. 2 presents comparative plots, showing the inhibition of serotoninuptake in intact freshly prepared human platelets effected by thepresence of NMC or citalopram, expressed in percent of inhibition of theuptake of [³H]serotonin as a function of the concentration of the testedcompounds.

As can be seen in FIG. 2 and Table 2, NMC was shown to inhibit [³H]5-HTuptake in freshly isolated human platelets with an inhibition constant(Ki) value of 52.0±15 nM, compared with 4.3±0.9 nM as determined forcitalopram.

The capacity of NMC to compete for [³H]citalopram binding and to inhibit[³H]5-HT uptake in human platelets indicate that NMC is a potentanti-platelet agent.

Assay of [³H]Citalopram Binding and [³H]5-HT Uptake in Rat Brain:

FIG. 3 presents comparative plots, showing the inhibition of the bindingof [³H]citalopram to the rat brain membranes serotonin transporter(5-HTT) by N-methyl-citalopram (NMC) or citalopram as a function of theconcentration of the competitive inhibitor. The Ki values in the 5-HTTbinding experiments in rat brain membranes, were 53±15 nM for NMC and3±1 nM for citalopram.

FIG. 4 presents comparative plots, showing the inhibition of[³H]serotonin uptake in freshly prepared rat brain synaptosomes effectedby the presence of the tested compounds, NMC and citalopram, expressedin percent of inhibition of the uptake of [³H]serotonin. The Ki valuesof the [³H]5-HT uptake experiments in rat brain synaptosomes, were44±6.6 nM for NMC and 4.5±0.7 nM for citalopram.

As can be seen in FIGS. 3 and 4, NMC exhibited about the same activityin rats as observed in the assay for human platelets (FIGS. 1 and 2).

Even if small amounts of NMC do penetrate the BBB during chronictreatment, its biological CNS activity is expected to be about 10-foldless potent for the CNS 5-HTT compared with citalopram, as shown by itsreduced affinity in competing for [³H]citalopram binding (almost 20-foldless) or to inhibit [³H]5-HT uptake (8-fold less) in the rat brain, ascompared with citalopram (see, FIGS. 3 and 4).

Measurements of ADP Induced Human Platelet Aggregation:

Serotonin, which is known to increase the platelet aggregation effectsof the classical platelet agonists ADP [24, 25], collagen [26] oradrenaline [27], was used in these experiments together with ADP toinduce platelet aggregation. This was employed to show that both testedcompounds, citalopram and N-methyl-citalopram (100 μM), can stronglyinhibit the aggregation of human platelets induced by 1.25 μM ADP in thepresence of 50 μM serotonin.

Platelet-rich plasma (PRP) samples were pre-incubated with the testedcompounds or PBS control as described hereinabove. FIG. 5 presentscomparative plots of the measurement of platelet aggregation as recordedfor a sample from a healthy donor, showing the percent inhibition ofhuman platelet aggregation effected by citalopram (plot denoted “Channel4” and colored in brown), by N-methyl-citalopram (plot denoted “Channel3” and colored in blue), and by the control PBS (plot denoted “Channel2” and colored in red).

FIG. 6 presents a repeat of the experiment presented in FIG. 5 asmeasured for a sample taken from the same healthy donor, showing thepercent inhibition of human platelet aggregation effected by citalopram(plot denoted “Channel 3” and colored in blue), by N-methyl-citalopram(plot denoted “Channel 2” and colored in red), and by the control PBS(plot denoted “Channel 1” and colored in green).

As can be seen in both FIGS. 5 and 6, the addition of ADP initiatesplatelet aggregation within about 20 seconds. For the PRP samplespre-incubated for 15 minutes with PBS as control, platelets aggregationhas reached a value of 85.1% or 91.8% of the maximal possibleaggregation, for measurements presented in FIGS. 5 and 6 respectively,during the period of 5 minutes observation by the aggregometer. For PRPsamples pre-incubated with citalopram (final concentration 100 μM) theaggregation had reached a value representing only 23.1% and 28.7%, formeasurements presented in FIGS. 5 and 6, respectively, of the maximumpossible aggregation. For PRP samples pre-incubated withN-methyl-citalopram (final concentration 100 μM) the aggregation hasreached a value representing only 20.2% and 22.9%, for measurementspresented in FIGS. 5 and 6, respectively, of the maximum possibleaggregation.

Both tested compounds exhibited strong inhibition of plateletaggregation.

Noted herein is that for both the tested compounds and the control PBS,there was a very similar early phase of platelet aggregation, which tookplace between about 40 seconds to 75 seconds following the addition ofADP. However, while for the PBS control there was a second phase ofplatelet aggregation, taking place from about 75 seconds to 300 secondsfollowing ADP addition, this second phase was entirely absent in the PRPsamples which were pre-incubated with citalopram or N-methyl-citalopram.

In particular, it can be seen that the tested compounds block the second(slower) phase of platelet aggregation. Furthermore, the data representinhibition in-vitro, however they were obtained with freshly isolatedhuman platelets of a healthy donor, and are the closest possibledemonstration in lieu of permission to give the tested compounds tohuman volunteers. Yet, considering that one of the tested compounds,citalopram, has been shown (among other SSRI drugs) to substantiallyreduce the risk of ischemic heart disease and MI in patients who take itchronically as discussed hereinabove, it is stated herein thatN-methyl-citalopram would be capable of inducing the same beneficialeffect in-vivo but without penetrating the brain, and therefore nothaving undesired CNS effects typical for SSRI drugs.

[³]Dopamine and [³H]Noradrenaline Uptake by Rat Brain Synaptosomes:

FIGS. 7A-B present a plot of [³H]dopamine uptake (FIG. 7A) and[³H]noradrenaline uptake (FIG. 7B) into rat brain synaptosomes as afunction of the concentration of a drug, comparing the inhibitory effectof citalopram, NMC and mazindol.

As can be seen in FIGS. 7A-B, mazindol, an inhibitor of dopamine andnoradrenaline uptake, inhibited [³H]dopamine uptake by 50% at 45 nM. Atthis concentration citalopram did not inhibit the uptake of dopaminewhile NMC exhibited less than 10% inhibition. The uptake of[³H]noradrenaline was inhibited by 50% at 8.5 nM mazindol whereas atthis concentration citalopram did not have any effect and NMC inhibitedless than 20% of the uptake.

[³H]Ketanserin Binding by Rat Brain Membranes:

The affinity of N-methyl-citalopram to rat brain membranes serotonin5-HT_(2A) receptors was evaluated in [³H]ketanserin binding assay. IC₅₀of the 5-HT_(2A) antagonist mianserin was 3.3 nM while up to 10 μM ofeither citalopram or NMC did not inhibit [³H]ketanserin (data notshown).

EXPERIMENTAL RESULTS In Vivo Studies

Determination of [3 μl]N-Methyl-Citalopram BBB Penetration In-Vivo:

The experiment was designed to show that N-methyl-citalopram does notpenetrate the mouse brain following intraperitoneal injection, asopposed to the uncharged counterpart, citalopram, using [³H]-labeledsamples thereof. The detected disintegrations per minute (DPM) values atthe different times after injection of either [³H]N-methyl-citalopram or[³H]citalopram are shown in FIG. 8.

FIG. 8 presents comparative plots of the accumulation of [³H]disintegration events per minute (DPM) in the cerebral cortex samples ofmice after the indicated times following the intraperitoneal injectionof either [³H]N-methyl-citalopram (marked in red circles) and[³H]citalopram (marked in black rectangles). The values represent themean+/−standard deviations (SD) for 3 mice for each compound, and for asingle mouse the time-point of 40 minutes. SD values for themeasurements in mice injected with N-methyl-citalopram were too small tobe shown on this scale.

As can be seen in FIG. 8, [³H]citalopram readily entered the brainshortly after its injection at the clinically relevant dose of 1.5 mgper kg body weight, reaching levels of about 3350 DPM per 500 μlcerebral cortex homogenate sample at 40 minutes, corresponding to about13,400 DPM for the entire cerebral cortex.

In sharp contrast, only very low amounts of radioactivity were detectedin the brain homogenate samples of mice following injections of asimilar dose (1.5 mg per kg body weight) of [³H]N-methyl-citalopram, ascan be clearly seen in FIG. 8. The radioactivity levels, expressed inDPM, in these brain homogenate samples were about 30-fold lower comparedwith the radioactivity levels in mice injected with [³H]citalopram.These DPM counts, ranging from 50 DPM at 5 minutes to 110 DPM at 40minutes were only slightly above the background radioactivity of about20 DPM.

Taking into consideration that the amount of radioactivity of injected[³H]N-methyl-citalopram was almost twice compared with that of[³H]citalopram, these data represent more than 50-fold less brainpenetration for [³H]N-methyl-citalopram compared with brain penetrationfor [³H]citalopram.

These results clearly show that [³H]N-methyl-citalopram does not, oronly minimally penetrate the mouse brain following intraperitonealinjection. Based on these results, it is estimated that the brainpenetration of N-methyl-citalopram following its intraperitonealinjection is about 50-fold less than that of citalopram. It is wellestablished that compounds behave similarly with respect to penetrationof the human or mouse brain following their administration to aperipheral organ. Hence, a compound that does not or only minimallyenter the mouse brain will show the same pattern for penetrating thehuman brain.

Determination of Bleeding Time in SSRI and Aspirin Treated Mice:

The working assumption in these experiments was that mice treated withaspirin or SSRIs are expected to be protected against the inducedpulmonary thromboembolism.

Tables 3, 4 and 5 present the results of the bleeding tests as describedhereinabove.

Table 3 presents data collected from chronically medicated mice havingtheir tail bleeding time measured after amputation of 3 mm from theirtail.

TABLE 3 Mean Experiment Dose Route of bleeding standard Mean No. Miceused (mg/kg) administration N time (sec.) deviation (n = 4) Control 1CJ57/black DDW p.o 4 97 45 179 2 CJ57/black DDW p.o 12 277 252 3CJ57/black DDW p.o 15 150 187 4 CJ57/black DDW p.o 16 157 193 5 ICR DDWor p.o/i.p 15 126 134 saline Aspirin 2 CJ57/black 40 p.o 12 389 228 2883 CJ57/black 40 p.o 14 230 225 4 CJ57/black 70 p.o 13 394 257 5 ICR 70p.o 14 145 186 Citalopram 1 CJ57/black 1.5 p.o 4 472 255 171 2CJ57/black 1.5 p.o 12 232 226 3 CJ57/black 2 p.o 14 179 221 4 CJ57/black4.5 p.o 14 115 149 5 ICR 10 i.p 12 66 23 NMC 1 CJ57/black 1.5 p.o 5 414265 191 2 CJ57/black 1.5 p.o 11 337 259 3 CJ57/black 2 p.o 15 150 187 4CJ57/black 4.5 p.o 15 108 78 5 ICR 10 i.p 13 124 144

Table 4 presents data collected from chronically medicated mice havingtheir tail bleeding time measured after amputation of 4 mm from theirtail.

TABLE 4 Mean Experiment Mice Dose Route of bleeding time standard No.used (mg/kg) administration N (sec.) deviation control 6 ICR DDW orp.o/i.p 23 218 194 saline aspirin 6 ICR 40 p.o 19 221 219 citalopram 6ICR 1.5 i.p 27 214 202 NMC 6 ICR 1.5 i.p 27 225 215

Table 5 presents data collected from chronically medicated mice (p.o.administration mode) having their tail bleeding time measured afteramputation of a segment corresponding to the area where tail diameter is2 mm at 1.5 mm depth into a tail restrainer.

TABLE 5 Mean Experi- bleeding ment Dose time standard No. Mice used(mg/kg) N (sec.) deviation control 1 CJ57/black DDW 10 174 165 aspirin 1CJ57/black 40 5 233 222 citalopram 1 CJ57/black 1.5 5 105 32 NMC 1CJ57/black 1.5 5 210 218

As can be seen in Tables 3-5, chronic treatment of mice with NMCresulted in a comparable effect on the increased bleeding as observedwith the other drugs used in the experiment. When all experiments werecombined, no significant differences were noted as compared to controlgroups. Furthermore, notable standard variation values were obtained inall groups representing a high variability of bleeding time betweenindividual mice in all groups. There is however an indication that lowdoses of citalopram or NMC (1.5 mg/kg/day) were more effective than thehigher doses (2 or 4.5 mg/kg/day). It is also noted that the oral (p.o.)administration of these drugs is more effective than i.p. injections inprolonging bleeding times.

Effect of SSRIs in Induced Pulmonary Thromboembolism in Mice:

Table 6 present percentage of mice who died or were paralyzed (loss ofrighting reflex for more than 30 seconds) after injection ofcollagen-epinephrine into tail vein. In Table 6, ^(a)p=0.016 and^(b)p=0.05 according to Mann-Whitney test versus control group.

As can be seen in Table 6, mice treated with aspirin were significantlyless prone to die or to be paralyzed after the injection ofcollagen-epinephrine mixture into tail vein. NMC administeredchronically to mice led also to a significant decrease in death orparalysis after the mixture injection.

Surprisingly, citalopram did not cause a significant improvementcompared to control group.

TABLE 6 Experiment Number of mice % Mean % death ± No, who died deathS.E.M Control 1 7/10 70 2 6/7 86 72 ± 2 3 8/13 61 Aspirin 1 5/10 50 25/12 42 40^(a) ± 7  3 3/11 27 Citalopram 1 7/9 78 2 8/13 61 70 ± 5 37/10 70 NMC 1 6/8 75 2 4/13 31  50^(b) ± 10 3 6/14 43Toxicology Preliminary Experiment:

Tables 7 and 8 present toxicology test results, showing mice reactionsfollowing i.p injections of citalopram (Table 7) or NMC (Table 8)observed over a period of three consecutive days.

TABLE 7 Mouse Mice behavior following citalopram injections Doseinjected number day 1 day 2 day 3 30 mg/day 1 normal slower movementsand slower movements reactions and reactions 2 normal slower movementsand slower movements reactions and reactions 50 mg/day 1 look quiet,look quiet, mobility looks quiet, mobility reduction mobility reductionreduction 2 look quiet, look quiet, mobility looks quiet, mobilityreduction mobility reduction reduction 100 mg/day  1 slower slowermovements and looks weak, movements reactions, looks weak andconvulsions then and reactions convulsions (weight recovered reduction)2 slower Weak and somnolent looks weak movements (weigh reduction) andreactions, apathetic

TABLE 8 Mice behavior Dose Mouse following NMC injections injectednumber day 1 day 2 day 3  30 mg/day 1 normal normal (weight normalreduction) 2 normal normal (weight normal reduction)  50 mg/day 1 normalnormal laid aside then recovered 2 look weak and difficulty laid asidethen lies aside to move recovered posterior legs 100 mg/day 1 laid asidethen laid aside then difficulty to recovered recovered move posteriorlegs, fast breathing, laid aside then recovered 2 Convulsions and death10 min after injection 3 laid aside then laid aside then recoveredrecovered

As can be seen in Tables 7 and 8, mice overdosed with NMC exhibitedadverse effects, yet, at less than 50 mg/kg NMC is not lethal to mice.In doses above 50 mg/kg of NMC can be toxic and a dose of 100 mg/kg canbe lethal.

The results presented above clearly show that NMC is a promisinganti-platelet drug, which does not cross the BBB and penetrate thebrain, and is therefore devoid of the adverse CNS influent ofcitalopram. These observations indicate that similar quaternary-nitrogenSRI compounds are likely to maintain their capacity for recognizing thehuman platelet 5-HTT and inhibiting its activity similarly to theirparent compounds in which the nitrogen atom is tertiary and whichtherefore do penetrate the BBB.

The in vivo results clearly demonstrated that NMC does not penetrate theBBB while exerting peripheral anti-clotting effect as high as otherknown drugs by protecting NMC-treated mice from acute pulmonarythromboembolism; and preliminary in vivo toxicology studies showed thatNMC is none-toxic at efficacious doses.

Therefore, NMC and similar quaternary SRI compounds have a clinicalpotential as anti-platelet agents (decreasing platelet aggregation)while being devoid of the undesired CNS effects of currently marketedSRI drugs, such as flattened emotions, reduced libido, andaggressiveness sometimes associated with current SRI compounds.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. A method of treating a disease or disorder inwhich reducing or preventing platelet aggregation and/orplatelet-endothelial interactions is beneficial, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a serotonin reuptake inhibitor (SRI) compound which comprisescitalopram modified so as to comprise at least one positively chargedgroup, said at least one positively charged group being a quaternaryammonium group, wherein said quaternary ammonium group has the formula:—(NR₁R₂R₃)⁺Z⁻ wherein: Z is an organic or inorganic anion; R₁ and R₂ areeach methyl; and R₃ is alkyl having from 1 to 4 carbon atoms.
 2. Themethod of claim 1, further comprising administering to the subject atherapeutically effective amount of an additional therapeutically activeagent.
 3. The method of claim 1, wherein R₃ is methyl.
 4. The method ofclaim 1, wherein said SRI compound is substantially incapable ofmodulating a serotonin level in the CNS of said subject.
 5. The methodof claim 4, wherein said reducing or preventing platelet aggregationand/or platelet-endothelial interactions is effected while substantiallymaintaining a level and/or activity of central serotonin, therebyavoiding CNS-related effects.
 6. The method of claim 1, wherein saiddisease or disorder is selected from the group consisting of acardiovascular disease or disorder, a cerebrovascular disease ordisorder, ischemic heart disease (IHD), myocardial infarction (MI),cerebral stroke, pulmonary embolism, type-2 diabetes-associated vascularabnormalities, pulmonary arterial hypertension, peripheral arterialocclusive disease, acute coronary syndrome, coronary artery disease(CAD) and coronary artery bypassing post-grafting.
 7. The method ofclaim 2, wherein said additional therapeutically active agent is ananti-platelet agent.